Endogenous adenosine can markedly potentiate the effects of insulin, in isolated adipocytes. This is thought to be mediated via A1 adenosine receptors, which are coupled to inhibition of adenylate cyclase (and possibly other effector systems) by the GTP-dependent regulatory protein, Gi. Prolonged incubation of adipocytes with the non-metabolizable adenosine analog, PIA (N6- phenylisopropyl adenosine) leads to down-regulation of adenosine receptors and Gi, and also causes a post-insulin binding defect in insulin action. Since the insulin resistance of conditions such as obesity and type II diabetes is caused, at least partly, by post- insulin binding defects these findings may be relevant to the pathogenesis of such conditions. The experiments in this proposal are designed to elucidate mechanisms of adenosine receptor and Gi regulation, to investigate the mechanism of PIA-induced insulin resistance, and to determine whether other components of the adipocyte adenylate cyclase system are altered after treatment of the cells with PIA. Adenosine receptor down-regulation will be studied by use of an 125I-labeled adenosine-protein conjugate, and by treating adipocytes with a protease to destroy cell-surface receptors. These techniques will determine whether endocytosis of adenosine receptors is involved in down-regulation. Use of a receptor photoaffinity probe will determine whether the receptors are structurally altered during down-regulation, or whether they are inactivated in the plasma membrane, and will allow further assessment of receptor internalization. The rate of Gi loss will be compared to that of adenosine receptor down-regulation as an indication of whether adenosine receptors and Gi are co- regulated. Subcellular fractionation will be performed to determine whether Gi is internalized in response to PIA. Two indexes of the presence of Gi in subcellular fractions will be utilized; 1) ability of GTP to inhibit adenosine receptor binding and 2) presence of the 41 kDa pertussis toxin substrate. The mechanism of PIA-induced insulin resistance will be investigated by determining whether 1) inactivation of Gi with pertussis toxin causes insulin resistance, 2) insulin receptor-associated protein kinase activity is decreased by PIA and 3) cyclic AMP levels are chronically increased in PIA-treated cells. To determine whether other components of the adenylate cyclase system are altered after PIA treatment, Gs will be assessed by cholera toxin- catalyzed ADP-ribosylation, and the catalytic subunit by forskolin-stimulated cyclase activity.